A Novel Strategy for Cell-Autonomous Gene Knockdown in Caenorhabditis elegans Defines a Cell-Specific Function for the G-Protein Subunit GOA-1

摘要

We developed a novel knockdown strategy to examine cell-specific gene function in Caenorhabditis elegans . In this strategy a null mutation in any gene is replaced with a genetically stable transgene that contains a wild-type copy of the gene fused to a 3′ tag that targets the mRNA transcript for degradation by the host nonsense-mediated decay (NMD) machinery. In NMD-defective animals, tagged transgene mRNA is expressed at levels similar to the endogenous gene it replaced and is translated into wild-type protein that fully rescues gene function. Cell-specific activation of NMD cell autonomously knocks down transgene expression in specific cell types without affecting its expression or function in other cells of the organism. To demonstrate the utility of this system, we replaced the [goa-1][1] gene, encoding the pan-neuronally expressed G-protein subunit [GOA-1][2], with a degradation-tagged transgene. We then knocked down expression of the transgene from only two neurons, the hermaphrodite-specific neurons (HSNs), and showed that [GOA-1][2] acts cell autonomously in the HSNs to inhibit egg-laying behavior. [1]: http://www.wormbase.org/db/get?name=goa-1;class=Gene [2]: http://www.wormbase.org/db/get?name=GOA-1;class=Gene